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The simultaneous detection of three kinds of small-molecule contaminants (antibiotics, mycotoxins, and hormones) in milk was realized by using an 8–17 DNAzyme-based fluorescent enzyme-linked immunosorbent assay (ELISA), in which 8–17 DNAzyme was utilized as the catalytic enzyme for amplifying the signal. Compared with the conventional ELISA in which horseradish peroxidase is used as the catalyzing factor, this 8–17 DNAzyme-based ELISA could achieve multicolor signal output with lower detection limits. The linearities for chloramphenicol, 17β-estradiol, and aflatoxin M1 were in the range of 0.3 ng/mL–3 μg/mL, 3 ng/mL–3 μg/mL, and 3 pg/mL–3 ng/mL with quantitation limits of 0.3, 3, and 0.003 ng/mL, respectively. This proposed scheme demonstrated that the 8–17 DNAzyme might be an effective substitute for horseradish peroxidase in ELISA for the development of ultrasensitive and multicolor fluorescence immunoassay, which would stimulate the development of ELISA in a new orientation.

Chemical pollutants such as heavy metals and antibiotics in the environment pose a huge threat to humans and animals. Our studies have demonstrated that the fluorescence of phycocyanin showed quenching responses towards both mercury (Hg2+) and ciprofloxacin (CIP), which acted in accordance with the “OR” molecular logic gate. In order to discriminate Hg2+ and CIP in application scenarios, cysteine (Cys) was utilized to design another “INHIBIT” logic gate, in which Hg2+ and Cys were the two inputs. Thus, an intelligent biosensor with dual-target identification capacity was successfully developed by using a fluorescent natural protein in an ingenious logic gate system.

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